Growth of a complex dendrite arbor is a prerequisite for neural circuit formation, and reduced arbor complexity in the cortex is a consistent finding in neurodevelopmental/neuropsychiatric disorders. For future therapeutic approaches to be conceivable, there is a critical need to elucidate the molecular mechanisms that regulate dendrite arborization. The ?-Protocadherins (?-Pcdhs), a family of 22 cell adhesion molecules encoded by the Pcdhg gene cluster, are required for normal cortical dendrite arborization; the mechanisms through which their diverse isoforms act are, however, poorly understood. The long-term goal is to identify molecular mechanisms that control neural circuit formation. The objective of this renewal application is to elucidate how both isoform diversity and common signaling mechanisms contribute to ?-Pcdh regulation of dendrite arborization. The central hypothesis is that ?-Pcdhs regulate arborization through: 1) diverse homophilic cell-cell interactions; 2) isoform-specific signaling via unique variable cytoplasmic domains (VCDs); and 3) shared signaling via a common C-terminal motif. Three Aims are proposed, each premised on published work from the current grant period, and each utilizing novel Pcdhg alleles generated through CRISPR/Cas9 genome editing. Aim 1: Establish the importance of a shared ?-Pcdh C-terminal motif in dendrite arborization in vivo. PKC phosphory- lation of a serine within a lysine-rich C-terminal motif abrogates its ability to bind phospholipids, and prevents ?- Pcdhs from inhibiting FAK and promoting dendrite arborization in vitro. To address the importance of this in vivo, 2 new mouse lines will be examined for arborization, ?-Pcdh protein stability, and FAK regulation: PcdhgS/A, in which the serine is mutated to an alanine, preventing phosphorylation; and PcdhgCTD, in which a premature stop codon results in proteins lacking the motif. Aim 2: Identify isoform-specific mechanisms through which the ?-Pcdh-C3 VCD regulates dendrite arborization. The VCD of the ?-Pcdh-C3 isoform uniquely inhibits Wnt signaling by binding to Axin1, a known regulator of dendrite arborization. Using in utero electroporation, Axin1 knockdown, and a novel PcdhgC3KO single-isoform knockout mouse, a distinct role for C3 in dendrite arborization will be ascertained. Aim 3: Ascertain the importance of ?-Pcdh isoform diversity for dendrite arborization and behavior. It's unknown whether isoform diversity generated by the endogenous Pcdhg gene cluster is important arborization. To test this in vivo, multiple novel mouse lines in which varying numbers of Pcdhg exons have been disrupted, reducing potential isoform diversity, will be analyzed. Additionally, anxiety- and memory-associated tests will be carried out on Pcdhg null, single-isoform overexpression, and reduced diversity mice to link cellular phenotypes to behavioral outcomes. The proposed research is innovative, because it utilizes CRISPR/Cas9 genome editing techniques to comprehensively assess the effects of altered ?-Pcdh repertoire and signaling in vivo. It is significant, because future therapeutic approaches will rely critically on identifying the molecules and signaling mechanisms that control dendrite arbor complexity.